Micro-lens systems for particle processing systems

ABSTRACT

The present disclosure provides improved optical systems for particle processing (e.g., cytometry including microfluidic based sorters, drop sorters, and/or cell purification) systems and methods. More particularly, the present disclosure provides advantageous micro-lens array optical detection assemblies for particle (e.g., cells, microscopic particles, etc.) processing systems and methods (e.g., for analyzing, sorting, processing, purifying, measuring, isolating, detecting, monitoring and/or enriching particles.

RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.16/259,311, filed on Jan. 28, 2019, which is a continuation of U.S.application Ser. No. 14/208,283, filed on Mar. 13, 2014, now U.S. Pat.No. 10,190,960, issued Jan. 29, 2019, which claims the benefit ofpriority to U.S. Provisional Application Ser. No. 61/784,323, titled“Micro-Lens Array Optical Detection for Particle Processing Systems andRelated Methods of Use,” filed on Mar. 14, 2013, the contents of all ofthe above applications being hereby incorporated by reference in theirentireties.

TECHNICAL FIELD

The present disclosure relates to optical systems for particleprocessing (e.g., cytometry including microfluidic based sorters, dropformation based sorters, and/or cell purification) systems and methodsand, more particularly, to micro-lens array optical assemblies forparticle (e.g., cells, microscopic particles, etc.) processing systemsand methods (e.g., for analyzing, sorting, processing, purifying,measuring, isolating, detecting, monitoring and/or enriching particles).

BACKGROUND

In general, particle processing (e.g., cytometry) systems (e.g.,cytometers) and methods are known. For example, some approaches toparticle processing or analyzing (e.g., cell purification) systems suchas sorting flow cytometers and other particle processing systems haveproven to be useful in life science research, industrial, diagnostics,and other medical applications.

In general, a cytometer can be described as a system that can measurelarge numbers of homogeneous and/or heterogeneous particle sets toachieve statistically relevant data sets that can be used to groupand/or identify subpopulations that reside within a given particlepopulation (e.g., within one or more samples). These measurements aresometimes performed optically (whether they are intrinsic or responsiveto an optical stimulus), or they may be electrical in nature (or someother physical, chemical, or biological characteristic) as a stream ofparticles passes through a measurement or inspection zone. The particlesets may include biological entities such as cells (e.g., bacteria,viruses, organelles, yeasts, spores, genetic material, spermatozoa, eggcells, multicellular organisms, etc.), or other organisms, or othernaturally occurring or synthetic/synthetically derived objects.

With the addition of sort functionality, a cytometer can also be used toisolate (e.g., physically separate) one or more particles of interestfrom a given sample through operator control. See, e.g., U.S. Pat. No.6,248,590, the entire contents of which is hereby incorporated byreference in its entirety. In general, this technique can be used toclassify and/or separate (e.g., purify or enrich) one or morepopulations as defined by the operator.

SUMMARY

According to certain aspects a particle processing system may include aparticle processing region and a detection region. The detection regionmay include a micro-lens array optical assembly. The particle processingregion, detection region and micro-lens array optical assembly areconfigured and adapted to process particles. The micro-lens arrayoptical assembly may include an aspheric lens system or an asphericmicro-lens array optical assembly. The micro-lens array optical assemblymay include one or more of the following features: common housing, atilt to the lens system to avoid blocking other light paths, a finelyground bevel to the lens array and/or housing, spectrally and/orspatially selective optical elements or optical filters within thehousing, isolation of optical paths, and/or optical collection fibersand/or detectors.

According to other aspects, a particle processing system may include adetection region including a micro-lens array and a particle processingregion configured to be removably and optically coupled to the detectionregion. The particle processing region may include a microfluidicchannel. The micro-lens array may include a micro-lens system associatedwith and in optical communication with the microfluidic channel. Themicro-lens system may include a single lens, multiple lenses(refractive, diffractive, Fresnel, gradient index (GRIN), reflectivemirrors, etc.) According to some embodiments, the micro-lens system mayinclude at least two aspheric lenses.

The particle processing system may further include a microfluidic chiphaving a plurality of particle interrogation regions and a plurality ofdetector assemblies configured to receive a fluorescence signal, anextinction signal and/or a scatter signal emitted from the particleinterrogation regions. A micro-lens array including a plurality ofmicro-lens systems may be provided. The particle processing system alsoinclude a receptacle for removably receiving the microfluidic chip,wherein the microfluidic chip has a plurality of microfluidic channels,and one or more light sources for illuminating the particleinterrogation regions.

The micro-lens array may be configured to collect fluorescence (orother) signals from the plurality of particle interrogation regions. Insome embodiments, the micro-lens array may have a non-zero workingdistance to the microfluidic chip. The micro-lens array may have aworking distance to the microfluidic chip ranging from approximately 50microns to approximately 50 mm. The working distance may include an airgap or other optically transmissive material. The micro-lens system mayinclude free space optics. The micro-lens system may include a pluralityof optical elements. A cross-section dimension of at least one of theoptical elements may be approximately equal to or greater than the widthof the micro-channel or the spacing between adjacent micro channels.

According to another aspect, a particle processing system may include anelectromagnetic radiation source; a signal detector assembly; amicrofluidic chip holder configured to removably receive a microfluidicchip having at least one particle interrogation region; and a micro-lenssystem configured to collect signals from the particle interrogationregion. The signal detector assembly may be at least one of afluorescence signal detector assembly, an extinction signal detectorassembly, or a scatter signal detector assembly. According to certainembodiments, the signal detector assembly may include a fluorescencedetector assembly and at least one of an extinction signal detectorassembly and a scatter signal detector assembly. According to certainembodiments, the micro-lens system may be configured to collectfluorescence signals from the particle interrogation region. Theparticle processing system may also include a fiber optic arrayconfigured to receive and transmit a signal to the extinction detectorassembly and/or the scatter detector assembly.

A micro-lens array having a plurality of the micro-lens systems may beprovided. Each micro-lens system may have a plurality of opticalelements. At least one of the optical elements may be an aspheric lens,a spherical lens, a reflective mirror array, or a gradient index lens.At least one of the optical elements may collect and collimate and/orfocus a fluorescence signal, a scatter signal, and/or an extinctionsignal. At least one of the optical elements may have a flat regionformed on a side surface. The flat region may be angled relative to theoptical centerline of the optical component. At least one of the opticalelements may be asymmetric with respect to the optical axis of theoptical element. At least one of the optical elements may have a firstsurface facing the particle interrogation region, the first surfacehaving an asymmetric cross-section.

According to some embodiments, at least one of the optical elements mayhave a diameter less than 1.0 mm. According to certain embodiments, atleast one of the optical elements may have a diameter less than 3.0 mm.According to other embodiments, at least one of the optical elements mayhave a diameter less than 25 mm.

According to certain aspects, each micro-lens system may collect afluorescence signal from a single micro channel. According to certainembodiments, each micro-lens system may collimate a fluorescence signalfrom a single micro channel. Additionally or alternatively, eachmicro-lens system may focus a fluorescence signal from a single microchannel. The micro-lens system may collect fluorescence signals from aplurality of interrogation regions.

According to other aspects, each micro-lens system may collect a lightscatter signal from a single micro channel. According to certainembodiments, each micro-lens system may collimate a scatter signal froma single micro channel. Additionally or alternatively, each micro-lenssystem may focus a scatter signal from a single micro channel. Themicro-lens system may collect scatter signals from a plurality ofinterrogation regions.

According to other aspects, an optical axis of the micro-lens systemsmay be oriented at an angle from the perpendicular to a micro channelprovided in the microfluidic chip. The angle from the perpendicular maybe up to approximately 60 degrees, and according to some embodiments,may range from approximately 5 to approximately 60 degrees.

According to various aspects, the micro-lens system may consist of onlyan aspheric lens; only two aspheric lenses; only an aspheric lens and afilter; or only two aspheric lenses and a filter. According to variousother aspects, the micro-lens system may consist of only a gradientindex lens; only two gradient index lenses; only a gradient index lensand a filter; or only two gradient index lenses and a filter. Accordingto even other aspects, the micro-lens system may consist of only aspherical lens; only two spherical lenses; only a spherical lens and afilter; or only two spherical lenses and a filter. According to stillother aspects, the micro-lens system may consist of only a reflectivemirror arrays; only two reflective mirror arrays; only a reflectivemirror array and a filter; or only two reflective mirror arrays and afilter.

According to certain aspects, the micro-lens system may include one ormore refractive and/or diffractive lenses. The micro-lens system mayinclude a spectral filter or spectral dispersing element. The micro-lenssystem may include one or more gradient index lens. The micro-lenssystem may include one or more reflective mirror elements. Themicro-lens system may include one or more spherical lenses. Themicro-lens system may include one or more cylindrical lenses. Themicro-lens system may include one or more spatial filters and/orspectral filters. The micro-lens system may include one or moremulti-element lenses. The micro-lens system may include any combinationof the above-noted elements.

According to yet another aspect, a micro-lens array may include a firstmounting portion have a first through hole configured to accommodate afirst lens for collecting and collimating a fluorescence signal andsecond mounting portion have a second through hole configured toaccommodate a second lens for collecting and focusing the collimatedfluorescence signal, and wherein the first and second mounting portionsare assembled with the first and second through holes aligned with oneanother. At least one of the first through hole and the second throughhole may be configured to accommodate a filter.

Any combination or permutation of embodiments is envisioned. Additionaladvantageous features, functions and applications of the disclosedsystems, assemblies and methods of the present disclosure will beapparent from the description which follows, particularly when read inconjunction with the appended figures.

BRIEF DESCRIPTION OF THE DRAWINGS

Exemplary embodiments of the present disclosure are further describedwith reference to the appended figures. It is to be noted that thevarious features and combinations of features described below andillustrated in the figures can be arranged and/organized differently toresult in embodiments which are still within the spirit and scope of thepresent disclosure. To assist those of ordinary skill in the art inmaking and using the disclosed systems, assemblies and methods,reference is made to the appended figures, wherein:

FIG. 1 schematically illustrates an exemplary particle processing systemaccording to the present disclosure;

FIG. 2 illustrates an exemplary microfluidic chip according to thepresent disclosure;

FIGS. 3A and 3B schematically illustrate exemplary particle processingsystems and micro-lens systems according to the present disclosure;

FIG. 4 schematically illustrates exemplary particle processing systemsand micro-lens systems according to the present disclosure;

FIGS. 5A and 5B illustrate exemplary optical elements according to thepresent disclosure;

FIG. 6 schematically illustrates exemplary particle processing systemsand micro-lens systems according to the present disclosure;

FIG. 7 illustrates exemplary optical elements according to the presentdisclosure;

FIGS. 8A, 9A and 10A schematically illustrate exemplary particleprocessing systems and micro-lens systems according to the presentdisclosure;

FIGS. 8B, 9B and 10B schematically illustrate variations of theexemplary particle processing systems and micro-lens systems of FIGS.8A, 9A and 10A;

FIG. 11 schematically illustrates exemplary particle processing systems,micro-lens systems and micro-lens arrays according to the presentdisclosure;

FIG. 12 schematically illustrates exemplary particle processing systems,micro-lens systems and micro-lens arrays according to the presentdisclosure;

FIG. 13 schematically illustrates exemplary particle processing systems,micro-lens systems and micro-lens arrays according to the presentdisclosure;

FIG. 14 illustrates a housing assembly for a micro-lens array (i) and anexemplary micro-lens array (ii) according to the present disclosure; and

FIG. 15 schematically illustrates exemplary particle processing systemsaccording to the present disclosure.

FIG. 16 schematically illustrates a (i) front view of the detector arrayand (ii) a side view of the detector array of FIG. 15.

In the description that follows, like parts are marked throughout thespecification and drawings with the same reference numerals,respectively. Drawing figures are not necessarily to scale and incertain views, parts may have been exaggerated for purposes of clarity.

DETAILED DESCRIPTION

The present disclosure provides improved optical systems for particleprocessing (e.g., cytometry including microfluidic based sorters, dropformation based sorters, and/or cell purification) systems and methods.More particularly, the present disclosure advantageously providesmicro-lens array optical assemblies for particle (e.g., cells,microscopic particles, etc.) processing systems and methods (e.g., foranalyzing, sorting, processing, purifying, measuring, isolating,detecting, monitoring and/or enriching particles), thereby providing asignificant commercial and/or operational advantage as a result.

The present disclosure provides advantageous optical systems forparticle processing systems and methods. More particularly, the presentdisclosure provides micro-lens array optical detection assemblies forparticle processing systems and methods (e.g., for analyzing, sorting,processing, purifying, measuring, isolating, detecting, monitoringand/or enriching particles).

In exemplary embodiments, a particle processing system for acquiringfluorescence signals, scatter signals (side and/or forward/backward),and/or extinction signals, etc. from a plurality of spatially separatedjets and/or channels includes one or more light sources for producing alight beam that passes through the jets and/or channels to be monitored,one or more micro-lens systems for capturing the light from the lightsource(s) after interaction with material (e.g., cells, particles and/orchemicals) in the jets and/or channels, and one or more detectors ordetector assemblies. The plurality of spatially separated jets and/orchannels may be supplied on one or more microfluidic chips or flowcells. The detectors, which may include light amplifying elements,typically detect each light signal and transduce the light signal intoan electronic signal. The electronic signals, typically representing theintensity of an optical signal, generally pass from the detector(s) toan electronic data acquisition system for analysis. In certainembodiments, the light amplifying element or elements may comprise anarray of phototubes, a multi-anode phototube, or a multichannelplate-based image intensifier coupled to an array of photodiodedetectors.

Some objectives of the present disclosure include providing one or moreof the following improvements over existing optical designs (e.g.,single optical axis design, large lens pair, etc.): 1) reducing opticalpath length; 2) reducing complexity (e.g., design and manufacture); 3)increasing optical collection efficiency and/or performance (e.g.,including reducing optical cross-talk between multiple flow measurementchannels); and/or 4) isolating optical paths of light collected from aplurality of objects (e.g., flow channels).

Moreover, the systems, assemblies and methods of the present disclosureadvantageously may include and provide, without limitation: 1) ensuringthat other light collection paths (e.g., light extinction and/orscatter) are not obstructed; 2) retaining a high numerical apertureand/or imaging performance, and/or 3) taking jet and/or channel width,chip and/or flow cell thickness and/or working distance into account.

A number of advantageous and design approaches were investigated for thesystems, assemblies and methods of the present disclosure, including,without limitation: 1) spherical lens arrays; 2) aspheric lens arrays;3) grin (gradient index) lens arrays; 4) mirror array collectionsystems; 5) bare fiber and other systems including lithography (e.g.,planar monolithic and stacked planar systems); and/or 6) otherreflective, diffractive and/or refractive approaches.

In certain aspects of the present disclosure, the particle processingsystems may utilize an aspheric lens array. Even more advantageously,according to certain exemplary embodiments, the particle processingsystems may include an aspheric micro-lens array optical assembly.

Further, in certain embodiments of the present disclosure, the opticalsystems for particle processing systems may include one or more of thefollowing: 1) a slight tilt to the lens system (e.g., to avoid blockingother light paths); 2) bevel to the lens array and/or to the lenselements themselves; 3) spectrally selective optical elements (e.g.,optical filter, spectral dispersion elements, etc.) within the housing;4) spatial and/or angularly selective elements; 5) isolation of opticalpaths, and/or 6) pinned or other high-precision locating features forassembling the components.

Embodiments include a micro-lens system, a micro-lens array, amicro-lens array for collecting and collimating light from a pluralityof micro channels associated with a plurality of flow cytometers, and amulti-channel microfluidic system including such micro-lens array.

An embodiment includes a micro-lens array for collecting and collimatinglight from a plurality of micro channels associated with a plurality offlow cytometers. The micro-lens array includes a plurality of micro-lenssystem disposed along the optical paths of the plurality of microchannels of the system. Further, the micro-lens array may include ahousing for mounting the plurality of micro-lens systems along theplurality of optical paths and for integrating the plurality ofmicro-lens systems with a fiber array. In some embodiments, the opticalsystem is configured for simultaneously collecting fluorescent lightemitted by a plurality of particles flowing in a plurality of microchannels.

The present disclosure provides a particle processing system including aparticle processing region and a detection region including a micro-lensarray optical assembly, wherein the particle processing region,detection region and micro-lens array optical assembly are configuredand adapted to process particles. The particle processing region may beprovided on microfluidic chips that are removably and interchangeablyconfigured for optical communication with the detection region and themicro-lens systems described herein.

Embodiments provide a micro-lens optical system for collecting signals(e.g., light) from a plurality of micro channels associated with aplurality of flow cytometers, and a multi-channel microfluidic systemincluding the optical system. Certain embodiments provide a micro-lensoptical system for collimating and/or focusing signals from a pluralityof micro channels associated with a plurality of flow cytometers. Thepresent invention is described below relative to illustrativeembodiments. Those skilled in the art will appreciate that the presentinvention may be implemented in a number of different applications andembodiments and is not specifically limited in its application to theparticular embodiments depicted herein.

The present disclosure also provides for a particle processing systemwherein the micro-lens array optical assembly includes an aspheric lensarray or an aspheric micro-lens array optical assembly. The presentdisclosure provides for a particle processing system wherein themicro-lens array optical detection assembly includes one or more of thefollowing features: a slight tilt to the lens system to avoid blockingother light paths, a bevel to the lens array and/or micro-lens system,spectrally selective optical elements or optical filters within thehousing, isolation of optical paths, and/or pinned or otherhigh-precision locating features for assembling the components.

The present disclosure is further described with respect to thefollowing examples; however, the scope of the disclosure is not limitedthereby. The following examples illustrate the systems and methods ofthe present disclosure of providing optical systems for particleprocessing (e.g., cytometry including microfluidic based sorters, dropsorters, and/or cell purification) systems and methods.

As used herein, the term particles includes, but is not limited to,cells (e.g., blood platelets, white blood cells, tumorous cells,embryonic cells, spermatozoa, etc.), synthetic beads (e.g.,polystyrene), organelles, and multi-cellular organisms. Particles mayinclude liposomes, proteoliposomes, yeast, bacteria, viruses, pollens,algae, or the like. Particles may also refer to non-biologicalparticles. For example, particles may include metals, minerals,polymeric substances, glasses, ceramics, composites, or the like.Additionally, particles may include cells, genetic material, RNA, DNA,fragments, proteins, etc. or beads with fluorochrome conjugatedantibodies.

As used herein, the term “microfluidic” refers to a system or device forhandling, processing, ejecting and/or analyzing a fluid sample includingat least one channel having microscale dimensions. The term “channel” asused herein refers to a pathway formed in or through a medium thatallows for movement of fluids, such as liquids and gases. The term“micro channel” refers to a channel, preferably formed in a microfluidicsystem or device, having cross-sectional dimensions in the range betweenabout 1.0 μm and about 2000 μm preferably between about 25 μm and about500 μm and most preferably between about 50 μm and about 300 μm. One ofordinary skill in the art will be able to determine an appropriatevolume and length of the channel for the desired application. The rangesabove are intended to include the above-recited values as upper or lowerlimits. The channel may have any selected cross-sectional shape orarrangement, non-limiting examples of which include a linear ornon-linear configuration, a U-shaped or D-shaped configuration, and/or arectangular, triangular, elliptical/oval, circular, square, ortrapezoidal geometry. A microfluidic device or chip may include anysuitable number of channels for transporting fluids. The microfluidicchip may include a disposable cartridge with a closed channel system ofcapillary size.

A microfluidic particle (e.g., cell) sorting system for a microfluidicchip, in accordance some embodiments, may have a wide variety ofapplications as a therapeutic medical device enabling cell-basedtherapies, such as blood transfusion, bone marrow transplants, and/ormobilized peripheral blood implants. Embodiments of microfluidic sortingsystems may be capable of selecting cells based on intrinsiccharacteristics as determined by interaction of light with the cells(e.g., scatter, reflection, and/or auto fluorescence) independent ofprotocols and necessary reagents. A microfluidic system may employ aclosed, sterile, disposable cartridge including a microfluidic chip. Themicrofluidic system may process particles (e.g., cells) at high speeds,and deliver particles (e.g., cells) with high yield and high purity.

Example 1: Microfluidic Flow Sorter Particle Processing System

Referring now to FIG. 1, a particle processing system 200 may beconfigured, dimensioned and adapted for analyzing, sorting, and/orprocessing (e.g., purifying, measuring, isolating, detecting, monitoringand/or enriching) particles (e.g., cells, microscopic particles, etc.)or the like. For example, system 200 may be a cytometer and/or a cellpurification system or the like, although the present disclosure is notlimited thereto. Rather, system 200 may take a variety of forms, and itis noted that the systems and methods described may be applied to otherparticle processing systems.

In exemplary embodiments, system 200 is a microfluidic flow sorterparticle processing system 200 (e.g., microfluidic chip based system) orthe like. Exemplary microfluidic flow sorter particle processing systemsand components or the like are disclosed, for example, in U.S. Pat. Nos.8,529,161 (Ser. No. 13/179,084); 8,277,764 (Ser. No. 11/295,183);8,123,044 (Ser. No. 11/800,469); 7,569,788 (Ser. No. 11/101,038);7,492,522 (Ser. No. 11/906,621) and 6,808,075 (Ser. No. 10/179,488); andUS Patent Publication Nos. 2012/0277902 (Ser. No. 13/342,756);2011/0196637 (Ser. No. 13/022,525) and 2009/0116005 (Ser. No.12/259,235); and U.S. Patent Applications Ser. Nos. 61/647,821 (Ser. No.13/896,213) and 61/702,114 (Ser. No. 14/029,485), the foregoing beingincorporated herein by reference in their entireties.

In further exemplary embodiments, system 200 is a multi-channel or multijet flow sorter particle processing system (e.g., multiple capillariesor multiple fluid jet-based systems) or the like. Exemplarymulti-channel or multi jet flow sorter particle processing systems andcomponents or the like are disclosed, for example, in US PatentPublication No. 2005/0112541 (Ser. No. 10/812,351), the entire contentsof which is hereby incorporated by reference in its entirety.

FIG. 1 illustrates a system 200 suitable for implementing anillustrative embodiment of the present disclosure. System 200 includes amicrofluidic assembly 220 (e.g., microfluidic chip). Microfluidicassembly 220 includes and/or is in communication with a particleinspection region and a sample fluid input region. Microfluidic assembly220 includes a plurality of channels for conveying a substance, such asparticles or cells, therethrough. In certain embodiments and as can beunderstood by those familiar in the art, microfluidic assembly 220 maybe a combination of cuvettes, capillaries, nozzles, or jets which maycombine to produce a multichannel particle processing system. Thechannels transport fluid and/or particles through the assembly 220 forprocessing, handling, and/or performing any suitable operation (e.g., ona liquid sample). Assembly 220 may include any suitable number of microchannels for transporting fluids through assembly 220. Alternatively, alesser number of channels than particle paths is envisaged whereby, forexample, multiple particle streams are injected into a singlemicrofluidic channel.

In exemplary embodiments, an optical detector system 226 for use withmicrofluidic assembly 220 is provided. At least a portion of opticaldetector system 226 may be implemented in a particle inspection regionconfigured for the interrogation of the particles in this region.

At least a portion of the optical detector system 226 may monitor flowthrough a plurality of channels simultaneously. In exemplaryembodiments, system 226 can inspect individual particles for one or moreparticular characteristics, such as size, form, fluorescence, opticalscattering, as well as other characteristics. It is noted that system226 is not limited for use in particle or cell sorting systems and maybe implemented in any suitable system having a substance, such asparticles, to be monitored within one or more channels.

System 200 also includes at least one electromagnetic radiation or lightsource 221 (e.g., a laser source or the like) for simultaneously orsequentially illuminating at least a portion of each of themicrofluidics channels 30 (e.g., an interrogation region 222) (see alsoFIG. 3). The electromagnetic radiation source 221 may be coupled toand/or in communication with beam shaping optics 225 (e.g., segmentedmirror/mirrors or the like) for producing and forming a beam ofelectromagnetic radiation (e.g., light) 227. The light source 221 may beprovide as one or more monochromatic light sources, polychromatic lightsources, or any combination of the aforementioned. As a non-limitingexample, the light source 221 may be an optical pumped semiconductor(OPS) laser device producing about 200 mW, 2 W, or 10 W at a wavelengthof 488 nm with minimal optical noise. As another non-limiting example, adiode pumped solid state (DPSS) laser may be used, which is capable ofgenerating different wavelengths of light, such as 355 nm at 300 mW or 2W, or 532 nm at 1 W, 2 W, 5 W, or 10 W for excitation and/orillumination. In general, the electromagnetic radiation source(s) 221may have any suitable wavelength and one skilled in the art willrecognize that any suitable light source(s) may be used.

In some embodiments, the one or more radiation beams 227 may passthrough an optical mask aligned with the plurality of particle-conveyingchannels in the microfluidic chip assembly 220. The optical mask maytake the form of an array of pinholes with each pinhole corresponding toa channel 30. The electromagnetic radiation admitted by the pinholessubsequently passes through the conveying channels themselves. Theportion of electromagnetic radiation beam 227 admitted to each channelvia one or more associated pinholes may intersect and/or interact withparticles that are conveyed through the channel to create opticalsignals.

Examples of optical signals that may be produced in optical particleanalysis, cytometry and/or sorting when a beam 227 intersects a particleinclude, without limitation, optical extinction, angle dependent opticalscatter (forward and/or side scatter) and fluorescence. Opticalextinction refers to the amount of electromagnetic radiation or lightthat a particle extinguishes, absorbs, or blocks. Angle dependentoptical scatter refers to the fraction of electromagnetic radiation thatis scattered or bent at each angle away from or toward the incidentelectromagnetic radiation beam. Fluorescent electromagnetic radiationmay be electromagnetic radiation that is absorbed and/or scattered bymolecules associated with a particle or cell and re-emitted at adifferent wavelength. In some instances, fluorescent detection may beperformed using intrinsically fluorescent molecules.

In exemplary embodiments, optical detector system 226 may include one ormore detector subsystems 230 to capture and observe the optical signalsgenerated by the intersection of electromagnetic radiation beam 227 witha particle in a channel. Detector subsystems 230 may include one or moreextinction detector assemblies 231 for capturing extinction signals, oneor more scatter detector assemblies 233 for capturing scatter signals,and one or more fluorescence detector assemblies 235 for capturingfluorescence signals. In a preferred embodiment, detector system 226 mayinclude at least one extinction detector assembly 231, at least onescatter detector assembly 233, and at least one fluorescence detectorassembly 235. Detector assemblies 231, 233, 235 may includephotomultipliers, photodiodes, cameras, or other suitable device(s).Further, detector assemblies 231, 233, 235 may include fiber optics orother waveguide-type optical transmission elements to direct the signalsto the sensor elements.

According to certain embodiments, a single detector subsystem 230 may beassociated with a plurality of microfluidic channels and thus, mayreceive signals (simultaneously, sequentially, overlapping,non-overlapping, etc.) from each of the plurality of channels. Thedetector assemblies 231, 233, 235 may be connected to electronics (notshown) to analyze the signals received from the detector assembliesand/or control one or more aspects of the particle sorting system 200.

Still referring to FIG. 1, each detector assembly 231, 233, 235 may beassociated with optical signal collecting and transmitting elements. Forexample, each detector assembly 231, 233, 235 may be associated with afiber optic array 232, 234, 236, respectively. Fiber optic arrays 232,234, 236 may extend between the image plane (and the interrogationregion 222) and the detectors 231, 233, 235 to convey signals todetector assemblies 231, 233, 235 for receiving and analyzing thesignals. The fiber optic arrays 232, 234, 236 may include optical fibercoupler-splitters. Additionally, one or more of the detector subsystems230 may include one or more lenses, filters, mirrors, and/or otheroptical elements to collect, shape, transmit, etc. the signal exitingthe interrogation region 222 and being received by the detectorassemblies 231, 233, 235. According to certain embodiments, there may bea one-to-one correspondence between an optical fiber and a micro channel30 for any specific signal (e.g., extinction, scatter, fluorescence).

According to certain embodiments and referring to FIG. 2, microfluidicassembly 220 may be configured as a microfluidic chip 20 and may includea substrate 21 having a plurality of channels 30 (e.g., micro channels)disposed therein. The channels 30 may be configured to transport fluidand/or particles through the microfluidic chip 20 for processing,handling, and/or performing any suitable operation on a liquid sample(e.g., a particle sorting system). For example, each micro channel 30may be a flow cytometer. The channels 30 may be arranged parallel toeach other.

As best shown in FIG. 2, the microfluidic chip 20 may include an inputregion 24 in which a sample containing particles (e.g., cells) are inputinto the microfluidic chip 20. The sample may be input through a firstside 28 of the microfluidic chip. Each micro channel has aninterrogation region 222 associated therewith. Particles in channels 30may be detected while flowing through the interrogation region 222. Atthe interrogation region 222, individual particles may be inspected ormeasured for a particular characteristic, such as size, form,orientation, fluorescence intensity, etc. Interrogation region 222 maybe illuminated through a second side 29 of the microfluidic chip (seeFIG. 2). The microfluidic chip 20 may have a plurality of channels 30(e.g., micro channels), with the interrogation regions 222 of theplurality of channels 30 distributed across a source area 22.

The micro channels 30 may be evenly spaced in the source area 22.According to certain embodiments, a centerline-to-centerline spacingbetween the channels 30 may range from 0.2 mm to 5.0 mm. Thecenterline-to-centerline spacing between the micro channels 30 may beless than 4.0 mm, less than 3.0 mm, or even less than 1.0 mm. Accordingto certain embodiments, the centerline-to-centerline spacing between themicro channels 30 may range from 2.5 mm to 3.0 mm. Advantageously, tominimize the footprint of the microfluidic chip 20, thecenterline-to-centerline spacing between the micro channels 30 may beless than 2.0 mm, less than 1.5 mm, or even less than 1.0 mm. Accordingto certain embodiments, the centerline-to-centerline spacing between themicro channels 30 may range from 0.7 mm to 1.2 mm.

The microfluidic chip 20 may be formed with one or more substratelayers. As best shown in FIG. 3A and 4, the microfluidic chip 20 may beformed with a first substrate layer 20 a with the micro channel 30 (and,thus, also the interrogation region 222) formed therein. The substrateof the microfluidic layer may be glass, PDMS, PMMA, COC, or any othersuitably transmissive material. The thickness of the first substratelayer 20 a may range from approximately 100 μm up to approximately 1000μm. In certain preferred embodiments, the thickness of substrate layer20 a may range from approximately 200 μm up to approximately 600 μm. Forexample, the thickness of substrate layer 20 a may be approximately 400μm. In other preferred embodiments, the thickness of substrate layer 20a may range from approximately 500 μm up to approximately 900 μm. By wayof non-limiting examples, the thickness of substrate layer 20 a may beapproximately 700 μm or approximately 750 μm. In certain embodiments,the microfluidic chip 20 may be formed with only two substrate layers 20a, 20 b.

Referring now to FIG. 2, microfluidic chip 20 may include twenty-fourchannels 30 flowing through the source area 22. One of ordinary skill inthe art will appreciate that microfluidic chip 20 may include morechannels or fewer channels flowing through the source area (e.g., asnon-limiting examples, 2, 4, 8, 24, 36, 72, 144, or 288 channels).According to some embodiments, when microfluidic chip 20 has twenty-fourmicro channels 30, the source area may have an overall length extendingacross the plurality of channels 30 ranging from 70 mm to 80 mm.

According to certain embodiments, each of the plurality of microchannels 30 may include a sorting mechanism for directing particlesflowing within the channel into various downstream channels. The sortingmechanisms may be located within one or more sorting regions 26 on themicrofluidic chip 20. Sorting may be accomplished through one or moresorting mechanisms, which may include but are not limited to: mechanicaldisplacement of the particle by deflecting a membrane with apiezoelectric actuator, pressure due to thermal actuators, optical forcetechniques, dielectric methods, and other suitable sort mechanisms ortechniques.

The particle processing system 200 may include a receptacle or holderfor removably receiving microfluidic chip 20. Further, the particleprocessing system 200 may include one or more stages for positioning themicrofluidic chip 20 relative to the optical detection system 226. Thestages may allow for movement (translation and/or rotation) of themicrofluidic chip 20 and/or movement of optical components, such as amicro-lens array 260.

According to certain aspects, a detector subsystem 230 may include oneor more micro-lens systems 250. A plurality of micro-lens systems 250may be provided as a micro-lens array 260.

According to certain embodiments, and referring back to FIG. 1 and nowalso to FIG. 3A, the optical signal collecting elements (e.g., 232, 234,236, 250) of detector assemblies 231, 233, 235 may be located on anopposite side of the microfluidic chip 20 (and of the interrogationregion 222) from the electromagnetic radiation source assembly 221.Advantageously, these optical signal collecting elements are positionedas close as possible to the interrogation region of the microfluidicchannel in order to receive the strongest signal. In certainembodiments, the optical signal collecting elements of opticalextinction detector assembly 231, for example, a fiber array 232associated with extinction detector assembly 231, may be placed directlyopposite the electromagnetic radiation source 221, and may be alignedwith the incident electromagnetic radiation path 227 for detectingoptical extinction. The optical signal collecting elements of opticalscatter detector assembly 233, for example, a fiber array 234 associatedwith scatter detector assembly 233, may be oriented substantiallyperpendicular to the path of the incident electromagnetic radiation beam227 in the plane formed by the incident light vector and themicrofluidic channel it intersects. Alternatively, a fiber array 234associated with a scatter detection assembly 234, if any, may beoriented from 10 to 70 degrees, and more typically from 20 to 50degrees) from the upper surface of the microfluidic chip substrate.

A fluorescence detector assembly 235 may capture optical signals fromparticle fluorescence emanating from interrogation region 222 of microchannel 30. In order to provide a strong fluorescence signal it isdesired to capture as many fluorescent photons as possible and imagethem onto detector assembly 235. However, the fluorescence signalemitted from the interrogation region may be dispersed, and an opticalsignal collecting system able to collect more fluorescent photons than afiber optic array may be desired. Thus, according to certain aspects,optical signal collecting elements for the fluorescence detectorassembly 235 may include a micro-lens system 250 for collecting afluorescence signal emitted from each microfluidic channel 30. Aplurality of micro-lens systems 250 may be assembled into a micro-lensarray 260 (see FIG. 1) for collecting the fluorescence signal emitted bya plurality of microfluidic channels 30. The micro-lens array 260 may beprovided as a subassembly within a single housing. Advantageously,micro-lens array 260 provides a compact, multi-optical axis system easyto handle and align.

The example embodiments described herein disclose micro-lens systems 250and/or micro-lens arrays 260 (refer to FIG. 1) in the context of afluorescence detector assembly 235. In general, the micro-lens systems250 and/or micro-lens arrays 260 may be associated with any signal thatis emitted from an interrogation region of a particle processing system.Thus, the micro-lens systems 250 and/or micro-lens arrays 260 may beprovided to collect fluorescence signals, side scatter signals,extinction and/or forward scatter signals, etc. and transmit thosesignals to the associated detector assemblies.

When a particle processing system 200 is configured to capture andanalyze extinction signals, scatter signals and/or fluorescence signalsemanating from a plurality of closely spaced micro channels 30, the areaabove the interrogation regions 222 may become very crowded with thecompeting optical signal collection systems. Referring to FIG. 3A, anelectromagnetic radiation source 221 may be located below microfluidicchip 20 and may direct an electromagnetic radiation beam 227 into aninterrogation region 222 of a micro channel 30. As shown, the incidentlight beam 227 may be provided at about a 45-degree angle relative tothe channel 30. Above the interrogation region 222, an optical fiber 232associated with extinction detector 231 may be oriented along the lineof the exiting radiation beam 227 and an optical fiber 234 associatedwith scatter detector assembly 233 may be oriented at a side scatterangle to the radiation beam 227 (i.e., typically within plus/minus 45degrees of the perpendicular to the radiation beam 227). The signalreceiving ends of these optical fibers 232, 234 may be positioned withinthe immediate vicinity of the interrogation region 222. For example, theends of the optical fibers 232, 234 may be positioned in contact withthe substrate of the microfluidic chip 20. According to certainembodiments, wherein the elements are closely packed, the ends of theoptical fibers 232, 234 may be positioned within 20 μm, 50 μm, 100 μm,150 μm, up to 200 μm, or even up to 1 mm from the surface of themicrofluidic chip 20. In a preferred embodiment, the ends of the opticalfibers 232, 234 may be positioned from 20 μm to 80 μm from the surfaceof the microfluidic chip 20. In other embodiments, one or more of theends of the optical fibers 232, 234 may be spaced from approximately 1mm to approximately 25 mm from the surface of the microfluidic chip 20.

Thus, when all three signals (extinction, scatter, and fluorescence) arecollected, the real estate available for the placement and orientationof a micro-lens system 250 for collecting and transmitting signals tothe fluorescence detector assembly 235 is defined by a fluorescencesignal sector delimited by the centerline of the extinction signal beingcollected on one side and the centerline of the scatter signal beingcollected on the other side. In some embodiments and referring to FIG.3A, the fluorescence signal sector 237 has an angle A of less than 90degrees. In other embodiments, the sector may encompass an angle A ofapproximately 90 degrees, or up to 100 degrees, up to 120 degrees, oreven up to 140 degrees. Even further, in order not to block any portionof the excitation and/or scatter signals, the real estate for placingand locating a micro-lens system 250 for collecting and transmittingsignals to a fluorescence detector assembly 235 is offset by the widthsof the beams transmitting the excitation and scatter signals. Forexample, referring to FIG. 4, a half-width dimension (½ B_(W)) for theextinction beam may range from approximately 100 μm to approximately 500μm, with a typical half-width dimension ranging from approximately 250μm to approximately 350 μm.

An exemplary micro-lens system 250 for collecting, collimating, and/orfocusing light from an interrogation region 222 associated with a microchannel 30 is illustrated in FIG. 3A. The micro-lens system 250 islocated between the optical fiber arrays 232, 234 of the extinction andscatter detection assemblies 231, 233. In this embodiment, central axis251 (e.g., the optical path) of the micro-lens system 250 is orientedperpendicular to the plane of the microfluidic chip 20. The micro-lenssystem 250 may include a plurality of optical elements 50 a, 50 bdisposed along an optical path 251 of the system. For clarity, amicro-lens mounting system has been omitted.

As shown, the micro-lens system 250 may include two optical elements 50a, 50 b. The plurality of optical elements 50 may include a first lens252 that collects light (i.e., electromagnetic radiation) from aninterrogation region 222 at or near the object plane 253 of themicro-lens system 250. The first lens 252 is located closest to theinterrogation region 222 and is positioned a working distance D_(W) awayfrom the top surface of the substrate 20 a. The plurality of opticalelements 50 may include a second lens 254 that images the light onto animage plane.

The first lens 252 may be an aspheric lens, as shown. The aspheric lensmay be a plano-convex, biconvex, convex/concave, etc. The second lens254 may also be an aspheric lens, as shown. Thus, as shown in FIG. 3A,micro-lens system 250 may include two aspheric lenses 252, 254. In otherembodiments, some or all of the lenses may be aspheric. In yet furtherembodiments, some or all of the lenses may be spherical, aspheric orcylindrical.

In some embodiments, the micro-lens system 250 may include a symmetricalarrangement of optical elements 50. Such a symmetrical arrangement mayreduce or eliminate aberrations (lateral chromatic aberration,longitudinal chromatic aberration, spherical aberration, distortion,coma, etc.). Thus, as shown in FIG. 3A, the first and second lenses 252,254 may be identical lenses placed in mirror opposition to each other.In other embodiments, the first optical element may be different thanthe second optical element.

In the embodiment of FIG. 3A, the plurality of optical elements 50 a, 50b includes first and second refractive lenses 252, 254. In otherembodiments, diffractive and/or reflective elements (e.g., diffractiveoptics, reflective optics) may be used instead refractive lenses. Evenfurther, the optical elements 50 forming a micro-lens system 250 mayinclude spatial filters, spectral filters, beam splitters, gratings,etc.

FIG. 3A also shows the behavior of the plurality of optical elements 50in the micro-lens system 250 using ray tracing. First optical element 50a may collect and may collimate the fluorescence signal. Second opticalelement 50 b may focus the signal received from the first opticalelement 252. In general, the micro-lens system 250 need not collimatethe fluorescence signal.

An air-gap may separate the first and second optical elements 50 a, 50b. In general, the length of the air-gap, if any, is not critical.According to other embodiments, an optically transmissive spacer (e.g.,glass, plastic, fluid) may be located between the optical elements 50.

In an exemplary micro-lens system 250, one or more of the opticalelements 50 along the optical path may have a diameter D (or othercross-sectional dimension) less than 3.0 mm. According to a preferredembodiment, one or more of the optical elements 50 may have a diameter Dbetween approximately 1.8 mm and approximately 2.7 mm. In otherembodiments, one or more of the optical elements 50 may have a diameterD between approximately 0.5 mm and approximately 5.0 mm. In even otherembodiments, one or more of the optical elements 50 may have a diameterD greater than approximately 5.0 mm. Larger or smaller diameter opticalelements 50 may be provided. Further, the diameters or othercross-sectional dimensions of the optical elements 50 need not be thesame.

According to preferred embodiments and still referring to FIG. 3A, themicro-lens system 250 may have a relatively short length Ls measured asa distance between the first surface of the first optical element 50 aalong the optical path 251 and the last surface of the last opticalelement 50 b. For example, the length Ls may range from approximately2.0 mm to approximately 25.0 mm. In a preferred embodiment, the lengthLs may range from approximately 5.0 mm to approximately 12.0 mm. As oneexample, the length Ls may be less than 12.0 mm, less than 10.0 mm oreven less than 8.0 mm. Longer or shorter micro-lens systems 250 may beprovided.

In the exemplary micro-lens system 250 of FIG. 3A, the first opticalelement 50 a disposed along the optical path may have an asphericalconvex surface facing the object plane 253. In the exemplary micro-lenssystem 250 of FIG. 3A, the last optical element 50 b may have anaspherical convex surface facing the image plane. For example, in themicro-lens system 250 of FIG. 3A, the lenses 252, 254 may each be aconvex aspheric lens. As another example, the lenses 252, 254 may eachbe a plano aspheric lens.

In general, the optical components 50 may include refractive optics,reflective optics, Fresnel optics, diffractive optics, or anycombination thereof, and lenses with other geometric profiles (e.g.,plano, convex, concave, aspheric, toroidal, spherical, cylindrical,etc.).

In preferred embodiments, most or all of the transmissive opticalelements 50 (e.g., lenses, filters, gratings) in the plurality ofoptical elements forming the micro-lens system 250 may include materialshaving relatively low auto-fluorescence. In some embodiments, each lensin the plurality of optical elements 50 may include a material having anauto-fluorescence within a range of about 200-times to about 2-timesless than BK7 glass. Other materials, including for example, plastics,which may be high fluorescence materials, may be used.

The micro-lens system 250 may have a combination of optical propertiesthat make it particularly well suited for applications involving thecollection and/or collimation of light from a micro channel associatedwith a flow cytometer. For example, a micro-lens system 250 may have arelatively high numerical aperture. In some embodiments, a micro-lenssystem 250 may have a numerical aperture two-to-three times higher thana photolighography micro-fabricated continuous array system. As anexample, a micro-lens system 250 may have a numerical aperture ofbetween 0.40 and 0.60. In certain embodiments, the micro-lens system mayhave a numerical aperture of approximately 0.50. Further, a micro-lenssystem 250 may allow longer working distances D_(W) with greaterdiameter D optical elements as compared to a micro-fabricated continuousarray system, thus enabling the use of standard microfluidic chipsystems.

According to some embodiments, the micro-lens system 250 may have anf-number (N) of less than about 2.0 (e.g., within a range of about 0.7and 1.2) for light from all portions in the interrogation region. Such alow f-number optical system may be particularly useful for low lightapplications, such as collecting light from fluorescence, luminescence,phosphorescence, scattered light, plasmonic emission, and/or Ramanemission.

A working distance D_(W) between the object plane 253 and the firstoptical element (e.g., lens 252) of the micro-lens system 250 may benon-zero. A non-zero working distance prevents the micro-lens system 250from physically contacting the microfluidic chip 218. Thus, themicro-lens system 250 is prevented from bending or otherwise deformingthe chip 218 and the chip 281 is prevented from deforming the micro-lenssystem 250 and thus possibly changing the optical alignment.

The working distance D_(W) between the object plane 253 and the firstoptical element (e.g., lens 252) of the micro-lens system 250 may beless than 25 mm. According to some embodiments, the working distance mayrange from approximately 0.05 mm to approximately 25 mm, fromapproximately 0.05 mm to approximately 20 mm, from approximately 0.05 mmto approximately 15 mm, from approximately 0.05 mm to approximately 10mm, or even less than approximately 5 mm. If the working distance is toogreat, compared to the diameter of the closest optical element (i.e.,the optical element collecting the signal) the signal intensity may betoo low. Thus, for an optical element having a diameter of less than 3.0mm, a working distance of less than 5.0 mm may be preferred. Similarly,a micro-lens having an f-number of approximately 1.0 may be preferred. Asmall working distance may be particularly useful in a flow cytometrysystem in which particles that emit or scatter light are separated fromthe optical system by at least fluid in the channel and/or a top surfaceof a microfluidic chip.

According to some embodiments, at least a portion of the workingdistance D_(W) may consist of an air gap. Further, an opticallytransmissive spacer may be located between the micro-lens system 250 andthe substrate forming the microfluidic chip 20. This spacer may assistin optimally positioning the micro-lens system 250 from theinterrogation region 222. The spacer may also assist in maximizing thecoupling efficiency of light from the interrogation region 222 to themicro-lens system 250.

The micro-lens system 250 may have approximately a one-to-onemagnification. The magnification of the optical system may fall within arange of about 0.5 to about 5. According to other examples, themagnification of the optical system may range up to 10 times or even upto 100 times. In some embodiments, a variation in the magnificationacross the lens system may fall within a range of +/−5%.

The micro-lens system 250 may have a relatively small depth of field. Insome embodiments, a depth of field of the optical system may be within arange of about +/−0.05 μm and about +/−1500 μm.

The point spread function of the micro-lens system 250 for variouspoints across the source area may be characterized through encircledenergy. For all light energy incident on an image plane from a pointsource located in the source area, the encircled energy of the lenssystem can be described as a percentage of the total energy incident onthe source plane that is encircled by a circle of a specified radiusaround the centroid of the distribution (e.g., the percentage of lightthat falls within a circle). In some embodiments, the micro-lens system250 may be configured such that at least 90% of the energy incident onthe image plane emitted by a point source in the source area isencircled by a radius that ranges from approximately 50 μm toapproximately 100 μm (or a diameter approximately 100 μm toapproximately 200 μm). The encircled energy radius for at least 90% ofthe incident energy of the optical system may fall within a range ofapproximately 5 μm to approximately 500 μm. According to even otherexamples, the encircled energy radius for at least 90% of the incidentenergy of the optical system may range from approximately 0.5 μm toapproximately 3 mm.

According to certain aspects and referring now to FIG. 4, a particleprocessing system 200 may include a micro-lens system 250 oriented at anangle B to the perpendicular to the plane of the microfluidic chip 20.In FIG. 4, the micro-lens system 250′ is shown oriented at 15 degreesfrom the perpendicular. Typically, a micro-lens system 250 may beoriented at any angle up to 25 degrees, up to 30 degrees, or even up to35 degrees from the perpendicular. In general, a micro-lens system 250may be oriented at any angle up to 60 degrees or even up to 70 degreesfrom the perpendicular. Orienting the micro-lens system 250 at an anglemay allow larger diameter micro-lens optical elements to be provided inthe available real estate and/or may allow the micro-lens system to bemoved closer to the interrogation region.

FIG. 4 illustrates that more than two optical elements 50 may beincluded in the micro-lens system 250′. For example, micro-lens system250′ may include optical elements 50 a′, 50 b, 50 c. Optical element 50a′ may be an aspheric lens 252′; optical element 50 b may also be anaspheric lens 254; and optical element 50 c may be a filter 256.

In the configuration shown in FIG. 4, filter 256 is a long pass filterlocated in the air-gap between the two aspheric lenses 252′, 254. A longpass filter may be used to filter out wavelengths of light associatedwith the light source 221 to increase the ratio of the fluorescencesignals to the illumination light from the particles or from the fluid.Other types of filters may be included in the micro-lens system 250′(e.g., band pass, notch, line, low pass, high pass, cutoff, multi-band,polarizer, holographic, spectrally dispersive, etc.).

Even other elements may be associated with one or more of the detectorsubsystems 230. For example, as shown in both FIG. 3A and FIG. 4, anaperture array 258 may be provided between the micro-lens system 250 andthe detector assembly 235. In some embodiments, an array of aperturesmay be positioned after one or more optical elements to suppressunwanted light or other spurious signals that originate outside of theobject plane.

Due to the tilted orientation of the micro-lens system 250′ one cornerof the lower surface of the first optical element 50 a′ is locatedcloser to the microfluidic chip 20 than the other corner. Spacingbetween this corner of closest approach and the upper surface of themicrofluidic chip 20 is defined as the edge working distance D_(W). Asthe tilt of the micro-lens system 250′ increases and as the diameter ofthe first optical element 50 a′ increases, the smaller the edge workingdistance D_(W) becomes. The smaller this edge working distance D_(W),the more the micro-lens system 250′ encroaches on the space availablefor the transmission of the side scatter signal.

In order to maximize the intensity of the fluorescence signal collected,it is desired to maximize the diameter of the first optical element 50 aand to minimize the edge working distance D_(W). In order to alsoprovide sufficient angular space for the side scatter signal, a distanced1 from the centerline CL of the optical element 50 a′ to the side 258of the optical element 50 a′ closest to the microfluidic chip 20 may beless than a distance d2 from the centerline CL of the optical element 50a′ to the side of the optical element 50 a′ farthest from themicrofluidic chip 20. This may best be seen in FIG. 5A.

Further, as shown in FIG. 5A, the distance d2 may equal a radius R ofthe optical element 50 a′. Thus, as one example, the top surface (ts) ofthe optical element 50 a′ may be provided with a fully circularcross-section having a radius R, while a bottom surface (bs) of opticalelement 50 a′ may be provided as a circular cross-section with a chord.The top surface (ts) of the optical element 50 a′ may have a largerperimeter than the bottom surface (bs).

Referring to FIG. 5A, in some embodiments, the side 258 of the opticalelement 50 a′ may be provided at an angle C relative to the centerlineCL of the optical element. The angle C may typically range fromapproximately 5 degrees to approximately 45 degrees.

In some embodiments, a lowermost corner 259 of an optical element 50 a′may be “removed,” so that relative to a straight-sided opticalcomponent, a chamfered or beveled optical element 50 a′ may be provided.This “removal” of material from a lower corner 259 of the opticalelement 50 a′ may be effected by grinding down, shaving away, slicingoff, dicing, truncating, or using any other method for removing materialfrom an optical element. The material may be “removed” from a standardoptical element. Optionally, this “removal” of a lower corner 259 of theoptical element 50 a′ may be provided by molding or forming the opticalelement 50 a′ with the angled edge 258.

According to some embodiments, the portion of the optical element 50 a′that is “removed” or not supplied may not significantly affect theintensity of the transmitted fluorescent signal as compared to anoptical element 50 not having the portion removed. As best shown byexamining the ray trace in FIG. 4, it can be seen that any portion ofthe fluorescent signal passing through the “removed” portion may notform part of the transmitted collimated signal. Thus, “removing” thisportion 259 of the optical element 50 a′ may not diminish (or may notsignificantly diminish) the intensity of the transmitted fluorescencesignal.

Even further, referring to FIG. 5B, it is understood that the opticalelement 50 a′ may have a chamfer, bevel or notch 258′ provided aroundany portion, including a minority or a majority, of the perimeter of thebottom surface (bs). Thus, for example, such a chamfer, bevel or notch258′ may be provided around the entire perimeter of the bottom surface,such that optical element 50 a′ is symmetric (and d1 will equal d2).According to this embodiment, the bottom surface (bs) of the opticalelement 50 a′ will have a smaller perimeter than the top surface (ts)(and both d1 and d2 will less than R).

The micro-lens system 250′ of FIG. 4 does not have a symmetric, mirrorimage arrangement of optical elements 50. Specifically, aspheric lens252′ is not identical to aspheric lens 254. Aspheric lens 254 issymmetrical with respect its centerline CL (which, in this embodiment,is coincident with the optical axis 251. In contrast, aspheric lens 252′is asymmetrical with respect to its centerline CL or with respect to theoptical axis 251.

FIG. 6 illustrates an embodiment of a particle processing system 200similar to the embodiment of FIG. 3, with the exception that themicro-lens system 250 of FIG. 3 has been replaced with a micro-lenssystem 250″. Micro-lens system 250″ includes a first optical element 50a″ and a second identical optical element 50 b″. The first and secondoptical elements 50 a″, 50 b″ are symmetrically, mirror-image asphericlens 252″, 254″ arranged along the optical path.

As best shown in FIG. 7, optical element 50 a″ is a truncated opticalelement, wherein a portion 259″ has been “diced off” parallel to thecenterline CL of the optical element 50 a. The dimension d1 is less thanthe dimension d2. Further, because the side 258″ is parallel to thecenterline CL, the perimeter of the top surface (ts) is equal to theperimeter of the bottom surface (bs). According to certain embodiments,the portion 259″ “diced off” (or not supplied) may be represented by achord (when the cross-section of the optical element 50 a is considered)having a chord height or rise of d3. The chord height d3 may range fromapproximately 10 percent to approximately 50 percent of the diameter Øof the non-truncated optical element 50 a.

FIG. 6 illustrates the ray trace of the micro-lens system 250″. Themicro-lens system 250″ may be oriented at any angle. Further, FIG. 6illustrates that the truncated side of the optical component 50 a″ maybe facing the extinction fiber assembly 232. Alternatively (not shown),the truncated side of the optical component 50 a″ may face the sidescatter fiber assembly 234.

According to other aspects, the micro-lens system 250 may includevarious optical elements 50. For example, as illustrated in FIG. 8A,micro-lens system 250 may include spherical lenses 352, 354 and a filter256 positioned therebetween. Spherical or ball lens 352 collects andcollimates a signal emitted from microfluidic chip 20 and spherical lens354 focuses the signal collimated by lens 352. According to oneembodiment, lenses 352 and 354 may have a diameter of 1.1 mm, anumerical aperture of 0.5 and a working distance of 0.62 mm. Accordingto another embodiment, lenses 352 and 354 may have a diameter of 2.5 mm,a numerical aperture of 0.5 and a focal length of 2.35 mm. In avariation, the spherical lenses 352, 354 may be provided ashemispherical lens or half-ball lenses.

As illustrated in FIG. 9A, micro-lens system 250 may include gradientindex (GRIN) lenses 452, 454 and a filter 256 positioned therebetween.In general, gradient index lenses have first and second plane opticalsurfaces and continuously changing refractive index across the diameterof the lens. According to one embodiment, lenses 452 and 454 may have anumerical aperture of 0.46 and a focal length of 2.88 mm. According toanother embodiment, lenses 452 and 454 may have a numerical aperture of0.47 and a focal length of −3.03 mm. A GRIN lens pair may have anumerical aperture of 0.44, a focal length of −3.03 mm, an objectdistance of 700 μm, and an image distance of 48 μm. According to anotherembodiment, a GRIN lens pair may have a numerical aperture of 0.44, afocal length of −2.27 mm, an object distance of 700 μm, and an imagedistance of 48 μm. According to some embodiments, the numericalapertures in the embodiments may range from approximately 0.4 toapproximately 0.7

As illustrated in FIG. 10A, micro-lens system 250 may include reflectiveoptical components 552, 554 and a filter 256 positioned therebetween.According to some embodiments, the reflective optical arrays may beSchwarzschild or other reflective optical systems. The reflectiveoptical components 552 may include a concave mirror 552 b having anopening formed at the center thereof and a convex mirror 552 a arrangedopposite to the opening of the concave mirror. The concave mirror mayhave an aspherical surface. The concave mirror 552 b collects thefluorescence signal emanating from the microfluidic chip 20 and reflectsit to the convex mirror 552. The convex mirror 552 b collimates thecollected signal and sends it through the central opening formed withthe concave mirror 552 b. According to one embodiment, reflectivecomponents 552 and 554 may have a numerical aperture of 0.4 to 0.7 and aworking distance of 0.05 mm to 25 mm.

Although certain exemplary micro-lens system 250 includes a firstoptical element 50 a for collecting and collimating light and a secondoptical element 50 b for focusing the light, in some embodiments, amicro-lens system 250′″ may only include the first optical element 50 a.Further, according to some aspects, a first set of optical elements maycollect light, but not collimate the light. Thus, according to someembodiments, a single lens may be used to collect and focus the signal.

FIG. 3B illustrates that, according to some embodiments, the micro-lenssystem 250′″ may include a single aspheric lens 252 and may include afilter 256 (not shown). FIG. 8B illustrates that, according to someembodiments, the micro-lens system 250′″ may include a single sphericallens 352 and may include a filter 256 (not shown). FIG. 9B illustratesthat, according to some embodiments, the micro-lens system 250′″ mayinclude a single gradient index lens 452 and a filter 256. FIG. 10Billustrates that, according to some embodiments, the micro-lens system250′″ may include a single reflective optical array 552 and may includea filter 256. According to certain other aspects, one or more additionaloptical systems (not shown) may be employed for focusing the collectedand/or collimated signal after the signal has been transmitted by themicro-lens system 250″.

FIG. 11 illustrates a micro-lens array 260 having a plurality ofmicro-lens systems 250 a, 250 b. For clarity, the mounting or housing ofthe micro-lens array 260 is not shown. Also, for clarity only thefluorescence detector assembly 235 is shown. Each micro-lens system 250is associated with the interrogation regions 222 of a plurality of microchannels 30 a, 30 b. Any number of micro-lens systems 250 and any numberof micro channels 30 may be provided. The plurality of micro-lenssystems 250 may transmit signals to a single fluorescence detectorassembly 235 (directly or via multiplexing). Alternatively, eachmicro-lens system 250 may transmit a signal to a dedicated detectorassembly 235 (not shown).

As shown in FIG. 11, the micro-lens system 250 a may include a pair ofidentical aspheric lenses 252, 254 with a filter, such as a long passfilter 256, positioned along the optical path in the air-gap between theaspheric lenses 252, 254. As shown, micro-lens system 250 b may beidentical to micro-lens system 250 a. Alternatively (not shown), themicro-lens array 260 may include micro-lens systems 250 that are notidentical. Any of the micro-lens systems 250 described herein may beincluded in the micro-lens array 260.

FIG. 12 schematically illustrates a micro-lens array 260 wherein one ormore micro-lens systems 250 c, 250 d may be associated with more thanone micro channel 30 (and thus, with more than one interrogation region222). For example, each micro-lens system 250 c, 250 d may includeoptical elements 50 a, 50 b, 50 c, etc. Optical elements 50 a and 50 bmay be aspheric lenses, wherein each lens may receive signals from aplurality of interrogation regions. Optical elements 50 c may befilters. For example, the optical elements 50 a, 50 b may have adiameter less than 3.0 mm and the micro channels may have acenterline-to-centerline spacing of 0.9 mm, so that each micro-lenssystem 250 may receive signals from three microchannels. In oneembodiment, a three-channel optical element may be an aspheric lenshaving a diameter of 2.7 mm, an approximate numerical aperture of 0.5and a focal length of 2.14 mm. In another embodiment (not shown), asix-channel optical element may include an aspheric lens having adiameter of 5.0 mm, an approximate numerical aperture of 0.5 and a focallength of 5.14 mm. Thus, a single micro-lens system 250 may collectsignals from a plurality of micro channels 30.

FIG. 13 schematically illustrates another aspect of a micro-lens array260. One or more of the micro-lens systems 250″″ may include a first setof optical elements 150 a and/or a second set of optical elements 150 b.Each set of optical elements 150 may include more than a single lens orother optical element 50. In the particular embodiment of FIG. 13, thefirst set of optical elements 150 a includes optical elements 50 a and50 e and the second set of optical elements 150 b includes opticalelement 50 b. Optical element 50 a may be a plano convex lens; opticalelement 50 e may be a positive achromatic; and optical element 50 b maybe a biconvex lens. In general, optical elements 50 need not be anyparticular micro-lens.

The first set of optical elements 150 a may collect the fluorescencesignal; the second set of optical elements 150 b may focus the signalcollected by the first set of optical elements 150 a. As illustrated,there may be provided a different number of elements in the first set ofoptical elements from the second set of optical elements. Optionally,the first and second sets of optical elements 150 may include the samenumber of optical elements. Further, the first and second sets ofoptical elements 150 may be provided with identical optical componentsand, even further, the first and second sets of optical elements 150 maybe symmetrically arranged as mirror images. FIG. 13(i) illustrates amicro-lens array 260 which has a plurality of micro-lens systems 250″″.FIG. 13(ii) illustrates a micro-lens system 250″″ orientated at an angleto the chip 20.

FIG. 14(i) illustrates a micro-lens array 260 having a plurality ofmicro-lens system 250 arranged in a linear array of twenty-fourmicro-lens systems 250. Any of the above-described micro-lens systemsmay be provided in the micro-lens array 260. As shown in FIG. 14(i), ahousing 264 for the micro-lens array 260 may include an optical fiber tomicro-lens array interface block 265. Thus, in some embodiments,micro-lens array 260 may include a plurality of optical fibers forreceiving a signal from the micro-lens systems 250 and transmitting thesignal to the one or more detectors. Each optical fiber may receive asignal from one microfluidic channel. Further, the housing 264 for themicro-lens array 260 may include means 266 to mount or couple themicro-lens array 260 to the remainder of the particle processing system200.

The housing 264 may be configured to maintain the alignment and spacingof the micro-lens systems 250 relative to each other and relative toother components of the particle processing system 200. For example, thehousing system 264 may include components for adjusting relativepositions between the interrogation regions 220/micro channels 30 of themicrofluidic chip 20 and/or between other components of a particleprocessing system 200. The housing 264 may provide slotted holds orother adjusting mechanisms to allow for adjustment of relative linearpositions and/or relative orientations between the micro-lens systems250 and the interrogation regions 222. The housing 264 for themicro-lens array 260 may also include spacers or standoffs for preciselylocating the micro-lens array 260 with respect to a microfluidic chip20.

Referring to FIG. 14(ii), the micro-lens array 260 may include aplurality of micro-lens systems 250′. Each micro-lens system 250′ may beidentically provided with three optical elements 50 a′, 50 b, 50 c. Theoptical elements may include: first and second aspheric lenses and afilter. Each micro-lens system 250′ is configured to be associated withan individual interrogation region 222 of a micro channel 30. For thisparticular embodiment, the center-to-center spacing S of the micro-lenssystems 250′ may be approximately 3 mm. The micro-lens array 260 mayhave an overall length L of approximately 75 mm, an overall width W ofapproximately 5 mm, and an overall height H of approximately 8 mm.

According to certain aspects, the array 260 may be assembled byencapsulating the plurality of micro-lens systems 250′ in a polymericsubstrate 262. As non-limiting examples, the polymeric substrate may bePDMS or other polymeric materials. A fluorescence signal from aninterrogation region may enter a first surface (s1) of the substrate,may be collected, may be collimated, may be filtered, and may be focusedby the micro-lens system 250′ and then may exit through a second surface(s2) of the substrate.

Referring to the end view, each micro-lens system 250′ may be orientedat an angle to the first surface (s1) of the substrate. For example, themicro-lens system 250′ may be oriented at an angle of 15 degrees from aperpendicular to the first surface (s1) of the substrate. When the firstsurface (s1) of the substrate 262 is positioned relative to themicrofluidic chip 20 in the source region 22, each micro-lens system 250may be automatically aligned at a 15 degree angle from the perpendicularto the individual interrogation regions.

Further, the micro-lens array 260 may be configured to allow passageand/or reduce the blocking of an extinction signal or a scatter signalfrom the interrogation regions 222. For example, substrate 262 mayinclude a chamfered region 268 to allow micro-lens the micro-lens system250 to be placed even closer to the interrogation regions 222 withoutblocking some or all of the side scatter signal. This chamfer 268 mayextend along the entire length of the array 260. Advantageously, thischamfer 268 of the substrate 262 may coincide with the chamfer 258 ofthe optical element 50 a′.

According to other aspects, the array 260 may be assembled by providingone or more mounting blocks having through holes configured forreceiving the individual optical elements 50 (or subassemblies of theindividual components). Thus, for example, a micro-lens array 260 mayinclude a first mounting portion have a first through hole configured toaccommodate a first optical element 50 and a second mounting portionhave a second through hole configured to accommodate a second opticalelement 50. For example, the through holes may be provided with steppedregions (i.e., shoulders) for seating the optical elements 50. Spacersmay be provided to properly maintain the optical elements 50 within themounting portions. The through holes may also be configured toaccommodate more than one optical component, for example, by includingshoulders of different diameters for seating optical elements havingdifferent diameters. Each mounting portion may maintain the alignmentand spacing of the optical elements 50 in the assembly relative to eachother. The first and second mounting portions may be assembled togetherwith the first and second through holes aligned with one another,thereby forming the micro-lens array 260. High-precision locatingfeatures (e.g., pins, grooves, etc.) may be used to precision align thefirst and second mounting portions.

Within the micro-lens array 260, the individual optical paths for themicro-lens systems are completely isolated from one another. Thus,optical crosstalk is prohibited between adjacent micro-lens systems.

Thus, it has been disclosed that an optical signal collection subsystemfor a detector assembly may include a micro-lens array 260. According tocertain aspects the present disclosure provides for a particleprocessing system wherein a micro-lens array optical assembly mayinclude an aspheric lens system or an aspheric micro-lens array opticalassembly. The present disclosure provides for a particle processingsystem wherein the micro-lens array optical assembly may include one ormore of the following features: a slight tilt to the lens system toavoid blocking other light paths, a finely ground bevel to the lensarray, spectrally selective optical elements or optical filters withinthe housing, isolation of optical paths, and/or pinned high-resolutionlocating features.

The micro-lens array 260 may have a combination of properties that makeit particularly well suited for applications involving the collectionand/or collimation of light from a plurality of micro channelsassociated with a flow cytometer. For example, a plurality of micro-lenssystems 250 may be provided, each having a relatively high numericalaperture. Additionally, a micro-lens array 260 with its discrete opticalelements optically isolated within a housing may reduce opticalcrosstalk as opposed to a continuous micro-fabricated lens array. Themicro-lens array 260 may simplify integration with the remainder of thedetector assembly and alignment with the microfluidic chip. Evenfurther, the compact assembly of the plurality of micro-lens systems 250may allow for a smaller overall particle processing system 200.

According to another aspect and referring to FIGS. 15 and 16, a particleprocessing system 300 may include a microfluidic assembly 220, a spatialfilter array 257, and a detector array 230′. Light from theinterrogation region 222 of the particle processing system 300 may beselectively filtered by a spatial filter array 257 located in betweenthe detector array 230′ and the microfluidic assembly 220. The opticalsignal for each interrogation region 222 may then by detected by eachdetector element 239 in the detector array 230′. The light collectionnumerical aperture of each detection element 239 in the system isdetermined by the dimensions of the detector and the working distance ofthe detector from the interrogation region 222 of the microfluidicsystem 220. The field of view and the numerical of the detector may bedetermine by the dimensions of the spatial filter 257 and the distanceof the spatial filter 257 from the interrogation region 222 of themicrofluidic system 220 and the detector elements 239. Multiple spatialfilters may be combined to match the detector array's collection angleand field of view relative to the interrogation region 222 of themicrofluidic chip 220.

Although the systems, assemblies and methods of the present disclosurehave been described with reference to exemplary embodiments thereof, thepresent disclosure is not limited to such exemplary embodiments and/orimplementations. Rather, the systems, assemblies and methods of thepresent disclosure are susceptible to many implementations andapplications, as will be readily apparent to persons skilled in the artfrom the disclosure hereof. The present disclosure expressly encompassessuch modifications, enhancements and/or variations of the disclosedembodiments. Since many changes could be made in the above constructionand many widely different embodiments of this disclosure could be madewithout departing from the scope thereof, it is intended that all mattercontained in the drawings and specification shall be interpreted asillustrative and not in a limiting sense. Additional modifications,changes, and substitutions are intended in the foregoing disclosure.Accordingly, it is appropriate that the appended claims be construedbroadly and in a manner consistent with the scope of the disclosure.

What is claimed is:
 1. A particle processing system comprising: adetection region including a micro-lens array, the micro-lens arrayincluding a first micro-lens system having a plurality of free-spaceoptical elements disposed along a central axis, the plurality offree-space optical elements including a first micro-lens and aspectrally-selective optical element in a common housing; and a particleprocessing region having a microfluidic channel, the particle processingregion configured to be removably and optically coupled to the detectionregion.